Abstract Previous studies investigating the neurobiology underlying the reinstatement of drug-seeking behavior, an animal model of relapse in drug-addicted individuals, have pointed to the medial prefrontal cortex as a critical node in this circuitry. In contrast, significantly less work has examined how different regions in the lateral prefrontal cortex influence reinstatement. In particular, the insular cortex, a region within the lateral prefrontal cortex, has been implicated in craving and relapse. Functional imaging studies suggest that drug-associated cues activate the insular cortex in individuals addicted to a variety of different drugs. In addition, damage or inactivation of this region disrupts nicotine addiction in both humans and animals. However, despite the evidence suggesting a role for this region in addiction to other drugs, few studies have investigated how different subregions of the insular cortex regulate cocaine-seeking behavior, particularly in animal models that allow for more precise neurobiological investigations into the mechanisms underlying its role in relapse/reinstatement. Our own now-published findings indicate that inactivation of the dorsal agranular insular cortex (AId) attenuates cued reinstatement but has no effect on cocaine-prime reinstatement in rats. However, the circuitry mechanisms underlying the role of the AId in the reinstatement of cocaine seeking remain unknown. Therefore, the present proposal will investigate the projections from the AId to different downstream regions to determine their role in cued reinstatement. Male and female Sprague-Dawley rats will undergo cocaine self-administration, followed by extinction training, prior to undergoing cued reinstatement testing. The proposed aim will determine whether chemogenetically inactivating either the AId?nucleus accumbens core or AId?basolateral amygdala pathway reduces lever pressing during cued reinstatement. The findings from this proposal will be instrumental in elucidating the neural circuits by which the AId is regulating cocaine-seeking behaviors.